Metal ions such as magnesium, copper, zinc, manganese, nickel, and iron are essential for plant growth, in processes that range from respiration to photosynthesis, but deleterious when present in excess amounts. Others such as cadmium, aluminum, and lead have no nutritional value and are toxic. When present in large amount in the soil, metals interfere with the uptake of essential ions, biosynthesis of chlorophyll and nucleic acids, and lipid metabolism, thus profoundly affecting plant growth and development (Ouariti et al. (1997) Phytochemistry 45:1343-1350; Dykema et al. (1999) Plant Mol Biol 41:139-150).
With the necessity to regulate metal ion uptake and achieve metal ion homeostasis, plants have evolved a series of metal transporters and various metal-binding polypeptides and proteins. Metallothioneins and phytochelatins are intracellular sulfur-rich low molecular weight polypeptides that chelate metal ions such as cadmium, zinc, copper, and mercury, and are thought to play a role in detoxification. More recently, a group of metal transporters, the ZIP gene family, was identified in plants (Guerinot (2000) Biochim Biophys Acta 1465:190-198). IRT1, the first ZIP gene to be identified, encodes a protein that is able to transport iron, zinc, manganese, and cadmium (Rogers et al. (2000) Proc Natl Acad Sci USA 97:12356-12360).
A novel class of polypeptides that are capable of being isoprenylated and binding metal ions such as copper, nickel, and zinc has also been recently discovered (Dykema et al. (1999) Plant Mol Biol 41:139-150). These proteins appear to be soluble, unlike most isoprenylated proteins which are membrane-associated. In terms of structure, they share the CXXC metal-binding motifs, and contain repetitive regions rich in the amino acids Pro, Lys, Asp, Glu, and Gly, predicted to form alpha-helices. Preceding the carboxyl-terminus is a flexible region of 30-70 amino acids enriched in the amino acids Pro, Ala, Tyr, and Gly, predicted to form turns (Dykema et al. (1999) Plant Mol Biol 41:139-150). The eight amino acids proximal to the carboxyl-terminal isoprenylation CaaX motif are highly conserved, with a consensus sequence of FSDENPNA (SEQ ID NO:21) followed by the CaaX motif (Dykema et al. (1999) Plant Mol Biol 41:139-150).
Phytochelatins are a class of posttranslationally synthesized peptides ((γ-Glu-Cys)n-Xaa polymers, where n is 2-11) whose synthesis from glutathione is promoted by heavy metals. They bind heavy metals such as cadmium with high affinity and localize themselves bound with the heavy metal ions to the cell vacuoles, thus playing a role in detoxification. Their synthesis is mediated by the enzyme phytochelatin synthase (γ-glutamylcysteine dipeptidyltranspeptidase, EC 2.3.2.15) using glutathione and related thiol tripeptides as substrate, via the net transfer of a γ-Glu-Cys unit from one thiol peptide to another or to a pre-existing phytochelatin molecule. Phytochelatin synthase is activated by heavy metals, a reflection more of the enzyme's requirement for glutathione-like peptides containing blocked thiol groups for activity, rather than direct metal binding to the enzyme (Vatamaniuk et al. (2000) J Biol Chem 275:31451-31459). Nucleic acid fragments encoding phytochelatin synthase have been isolated from Arabidopsis, yeast, wheat, and worm (Ha et al. (1999) Plant Cell 11:1153-1163; Clemens et al. (1999) EMBO J. 18:3325-3333; Vatamaniuk et al. (1999) Proc Natl Acad Sci USA 96:7110-7115; Vatamaniuk et al. (2000) J Biol Chem 275:31451-31459; Vatamaniuk et al. (2001) J Biol Chem 276:20817-20820).
It is apparent that by manipulating phytochelatin synthase expression, phytochelatin levels may be controlled, and consequently, heavy metal detoxification and tolerance by plants as well. Increasing phytochelatin synthase expression may lead to increased heavy (transition) metal resistance which has a potential use as a selectable marker system for plant transformation studies. Also, plants may be engineered to grow in toxic metal rich soils or to remove pollutant metals from the environment through manipulating expression of phytochelatin synthase. Accordingly, the instant specification discloses nucleotide sequences encoding phytochelatin synthase which may be used for the above mentioned applications.